Epidemiology, Biochemistry and Evolution of Trypanosoma cruzi Lineages Based on Ribosomal RNA Sequences

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1 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I: 59-64, Epidemiology, Biochemistry and Evolution of Trypanosoma cruzi Lineages Based on Ribosomal RNA Sequences Bianca Zingales/ +, Beatriz S Stolf, Ricardo P Souto/ ++, Octavio Fernandes*, Marcelo RS Briones** Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 6077, São Paulo, SP, Brasil *Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil **Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil Key words: Trypanosoma cruzi - major lineages - ribosomal RNA - molecular epidemiology - phylogeny A puzzle in Chagas disease research has been to correlate molecular markers with epidemiology since isolates of Trypanosoma cruzi exhibit a broad host range, induce distinct clinical presentations in patients and show great diversity in biological and biochemical characteristics. Early studies on population genetics revealed substantial isozymic variability among isolates of T. cruzi defining three major groups or zymodemes named Z, Z and Z3 (Miles et al. 978, 980). Further analysis of 5 gene loci disclosed a greater heterogeneity determining the distribution of T. cruzi isolates into 43 zymodemes that could not be grouped by the authors in few natural clusters (Tibayrenc & Ayala 988). The main conclusion of these studies was that the population structure of this parasite is clonal rather than sexual, and, as a consequence, the present genetic and biological variability of T. cruzi is resultant from the independent evolution of clonal lines (Tibayrenc & Ayala 988). Acceptance of clonal propagation of T. cruzi implies that the classical definition of species is not fully adequate to this parasite. It has been proposed that each distinct genotype should be studied individually and special attention given to the most represented ones (Tibayrenc 995). On the other hand, other groups still believe that it is worth searching for genetically and ecologically coherent group- This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) and Conselho Nacional de Pesquisa (CNPq). + Corresponding author. Fax: bszodnas@quim.iq.usp.br ++ Present address: Department of Biochemistry, Boston University School of Medicine, 80 East Concord St., Boston, MA 08, USA Received 9 June 999 Accepted 9 August 999 ing of isolates (Macedo & Pena 998). Indeed the remarkable genetic diversity of T. cruzi (Morel et al. 980, Henriksson et al. 990, Macedo et al. 99) initially identified could be somewhat organized (Henriksson et al. 993, Tibayrenc 995). However, analyses of these fast evolving genetic markers could not clearly show defined clusters. TYPING OF T. CRUZI ISOLATES BASED ON A RRNA SEQUENCE In contrast to the diversity suggested by former approaches, analyses of markers with lower evolutionary rate such as multigenic families suggested dichotomy within T. cruzi isolates. PCR amplification of a sequence from the D7 divergent domain of the 4Sa rrna (LSU) gene indicated dimorphism among T. cruzi isolates and allowed the division of 6 parasite stocks into two groups (Souto & Zingales 993). This observation was confirmed by others following riboprinting analysis in 8 isolates from North America (Clark & Pung 994). Subsequently, a variable spot in the spacer region of the mini-exon gene was found in strains of T. cruzi which also showed dimorphism after PCR amplification (Souto et al. 996). The typing approaches based on rrna and mini-exon sequences were used to analyse 88 T. cruzi stocks from s, insects and sylvatic animals from several Latin America countries. Amplification of these sequences originated 5 or 0bp products for rdna and 300 or 350bp products for the mini-exon. Within individual isolates, one of the three associations was observed: the 5bp rdna product with the 300bp mini-exon product (defined as group ), the 0bp rdna product with the 350bp mini-exon product (defined as group ), and the presence of both rdna amplification products with the mini-exon group product (defined as group /) (Fig. ) (Souto et al. 996). RAPD analysis showed variability between individual isolates, however, tree analysis clearly indicated the presence of two major

2 60 T. cruzi Lineages Based on Ribosomal RNA Sequence Bianca Zingales et al. branches. Interestingly, the group isolates correlated precisely with one branch of the tree, and group and group / isolates correlated with the other branch (Fig. ) (Souto et al. 996). Our studies clearly indicate that T. cruzi isolates can be partitioned into two major Lineages, which we named Lineage and (Fig. ) (Souto et al. 996). In that study several isolates classified as Z and Z were analyzed and it was concluded that Z stocks belong to Lineage, while stocks classified as Z fall into Lineage. The position of Z3 was further investigated based on mini-exon sequence analysis and it was concluded that it constitutes a discrete sub Lineage within Lineage (Fernandes et al. 998a). This observation agrees with previous conclusions indicating that Z3 although distinct from, is related to Z (Miles et al. 980). The existence of these lineages can be noticed by detailed isoenzyme data (Tibayrenc 995). Concerning the major clones defined by Tibayrenc and Ayala (988), our typing assays indicate that clones 9/0 belong to Lineage, while clones 3 and 39 belong to Lineage. It should be pointed out that clone 39 maintains heterozygosity at several loci and is classified as group / (Souto et al. 996). More recently, the bi-polar grouping of T. cruzi has been supported by riboprinting analysis (Stothard et al. 998), rrna and mini-exon (spliced-leader) promoter sequences and activities (Floeter-Winter et al. 997, Nunes et al. 997a, b), microsatellite markers (Oliveira et al. 998) and structure of ribosomal spacers (Fernandes et al. 999a). Presently, several laboratories are involved in the definition of the relevance of the two lineages and their sub-groups with respect to the epidemiological and biological properties of T. cruzi. These studies as well as the analysis of the evolutionary origin of the two Lineages should indicate whether each Lineage constitutes, according to the modern concept of species, an ecological and a genetic unit (Macedo & Pena 998). THE RIBOSOMAL RNA CISTRON Southern blot analysis (Souto et al. 996), riboprinting (Clark & Pung 994, Stothard et al. 998) and restriction profiles of internal transcribed spacers (ITS) from the rdna cistron (Fernandes et al. 999a) clearly indicate that the overall structure of the ribosomal RNA cistron differs between isolates of Lineage and Lineage, supporting this division. It has also been shown that group / isolates have both types of rrna cistrons, with eight to ten-fold greater copy number of group - rdna genes relative to group -rdna genes (Souto et al. 996). Previous studies in different organisms have shown that rrna gene promoters exhibit speciesspecific control (Sollner-Webb & Towers 986). A plasmid construct bearing the promoter sequence from the rrna cistron of the CL strain drove the expression of chloramphenicol acetyl transferase (CAT) when transfected into epimastigotes of some T. cruzi strains, but surprisingly, was inactive in others (Tyler-Cross et al. 995). We examined the activity of the same construction in isolates belonging to the two major phylogenetic lineages. It was observed that CL strain (Lineage ) rrna promoter drove high CAT activity in Lineage isolates, but essentially no activity in Lineage strains. Thus, the CL rrna promoter exhibited a clear Lineage -specificity (Floeter-Winter et al. 997, Nunes et al. 997a). In order to analyse the activity of Lineage rrna promoter, we cloned the corresponding segment from the Dm8 strain. Alignment of this sequence (800bp) with rrna promoters from other strains of T. cruzi indicated 98% and 8% identity with sequences of Lineage and Lineage strains, respectively (Floeter-Winter et al. 997, Stolf 999). These data indicate that the sequence homology in this region also reflects the division into two lineages. We have also analysed the rrna promoter sequences from group / isolates, which contains two types of ribosomal RNA cistrons. It was observed that the type promoter sequence of NR cl3 clone (group /) has 96% identity with that of CL Brener clone (Lineage, group ) and 80% identity with that of Dm8 strain (Lineage, group ). On the other hand, the type promoter sequence of NR cl3 clone has 8% identity with that of CL Brener clone and 90% identity with that of Dm8 strain (Stolf 999). The activity of Lineage and Lineage promoters was investigated by transient expression of CAT reporter gene in different T. cruzi isolates typed as rdna group, group and group / (cf in Fig. ). It was observed that Lineage promoter is expressed in both group and group / strains, but shows no activity in group isolates. On the other hand, Lineage promoter is expressed in the three groups of isolates (Nunes et al. 997a, Stolf 999). In the specific case of group / it was observed that the expression of CAT driven by L promoter was higher than that driven by the L promoter. Interestingly, RT-PCR experiments conducted with total RNA extracted from group / isolates allowed to conclude that only the rrna cistron of type is transcribed in vivo (Stolf 999). Taken together, our data indicate that there are clear differences in the sequence and activity of the rrna promoter regions in the representatives of the two major lineages. The specificity of the promoter activity is not mutually exclusive, since group and group / strains express promoters

3 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I, LINEAGE CL B67 CA B47 SC43 cl Bug NR cl3 SO3 cl5 E sm e r. Y Basileu A ME rdna HOST / / / / / / / LINEAGE G Dm8 Tulahuen Silvio YuYu opossum opossum opossum Fig. : UPGMA tree based on the proportion of bands not shared among the isolates. For each isolate the corresponding rdna and mini-exon groups as well as the host from which it was derived are indicated (after Souto et al. 996). from both groups, but group strains only express group promoters. The reasons for these observations are unclear but might be related to the characteristics of the components of the transcription machinery in the T. cruzi groups. Nevertheless, since it has previously been shown for many eukaryotes that the rrna promoter activity is species-selective (Sollner-Web & Towers 986) the observation that T. cruzi rrna promoters show group-specificities supports the conclusion that these groups could be considered discrete taxa. EPIDEMIOLOGICAL DISTRIBUTION We have analysed by the 4Sa rrna and/or mini-exon typing methods more than 00 isolates from triatomine species and mammalian hosts from the domestic and sylvatic cycles originating from Brazilian states. Data provide evidence for a strong association of T. cruzi Lineage with the domestic cycle, while Lineage is preferentially encountered in the sylvatic cycle (Fernandes et al. 998b, 999b, Zingales et al. 998, cf also Fig. ). Data also suggest that Lineage has properties that favour infections, possibly due to higher parasitemia, since all parasites isolated from seropositive individuals from endemic regions belong to this Lineage (Fernandes et al. 998b, Zingales et al. 998). On the other hand, Lineage was isolated from very few seropositive cases in Amazonas where T. cruzi is enzootic (Zingales et al. 998). These observations concerning the general behaviour of the two lineages in chagasic patients in Brazil agree with data regarding the distribution of two major clones: 0 (Lineage ) and 39 (Lineage ) in children and vectors from a Bolivian endemic area (Breniere et al. 998). It was concluded that clone 39 is prevalent in patients, while in vectors (Triatoma infestans) clones 0 and 39 are found with comparable frequencies. These results suggest a limited selection in the transmission of the two clones and a drastic control of clone 0 parasitemia by the immune system of children patients. Analysis of 68 T. cruzi stocks from mammals and triatomines of the Atlantic Coast rainforest in the State of Rio de Janeiro suggests a preferential adaptation of Lineage to primates and Lineage to opossums (Fernandes et al. 999b), as has also been concluded from studies conducted in Georgia State (USA) (Clark & Pung 994, Pung et al. 998).

4 6 T. cruzi Lineages Based on Ribosomal RNA Sequence Bianca Zingales et al. BIOLOGICAL EVIDENCE Several biological evidence also support the conclusion that the two phylogenetic lineages have different characteristics. Experimental infection of marsupials with strains of Lineages and indicates the elimination of type strain from the host, while the type strain promotes a permanent and very mild infection (Carreira et al. 996). In vivo and in vitro experiments suggest that the two lineages differ markedly in infectivity to mammals. In fact, it has been shown that two T. cruzi strains Y and CL (Lineage ) are highly infective to mice, while strain G (Lineage ) does not produce parasitemia (Yoshida 983). This observation agrees with data derived from the analysis of isolates from chagasic patients (Fernandes et al. 998b, Zingales et al. 998). It has also been reported that Lineage strains are four times more infective to HeLa cells than Lineage strains. These results have been correlated with the expression of glycoprotein gp90 which seems to inhibit calcium mobilization required for cell invasion and is present in the eight analyzed strains of Lineage but not in Lineage isolates (Ruiz et al. 998). Regarding the parasite-triatomine vectors interaction, it is known that growth and metacyclogenesis of different T. cruzi isolates in the insect depend on the strain of the parasite (Garcia & Azambuja 99, Lana et al. 998). For example, it has been shown that infection of Dipetalogaster maximus with two different clones of T. cruzi showed that only one clone underwent metacyclogenesis. Similar results were seen when different strains and clones of T. cruzi infected Rhodnius prolixus. It was also observed that clone Dm8c (Lineage ) was two to three orders of magnitude more resistant to a lytic agent purified from the crop of R. prolixus than the Y strain (Lineage ). Resistence to lytic activity, as well as reactivity to specific lectins found in the digestive tract of triatomines may provide selective advantages for the development of certain strains of T. cruzi over other strains (cf. Garcia & Azambuja 99). EVOLUTION OF T. CRUZI LINEAGES Comparative sequence analysis of the variable region D7 within the LSU rrna was used to check whether the divergence between T. cruzi Lineages and occurred before the divergence of the extant strains as suggested by RAPD (Souto et al. 996) and also to verify whether the lineages are truly monophyletic. Therefore, sequences of the D7 region were determined for T. rangeli and 4 strains of T. cruzi and aligned. This alignment was used to build maximum likelihood trees using different transition models and rooted by outgroup (T. rangeli). The tree presented suggests that the divergence between T. cruzi Lineages and occurred before the divergence of the extant strains and separate T. cruzi into two groups in 00% of bootstrap replicates (Fig. ). Also a third group seems to be present which corresponds to group /, where the presence of two types of rdna cistrons has been shown. Alignment of the sequence of type D7 region of SO3 (group /) places this isolate among Lineage strains (Fig. ) while type D7 sequence does not allow the establishment of a clear position in the tree. In fact, depending on the algorithm parameters used (parsimony, different models of maximum likelihood and gamma distribution), group / in some trees seems to be more related to Lineage and in other trees to Lineage. Sequence comparisons of small subunit rdna (SSU) and faster evolving genes will probably help to determine whether group / is more closely related to Lineage or or if it really is a third lineage of strains. T. rangeli Fig. : phylogeny of several Trypanosoma cruzi isolates based on D7 region of the LSU rdna. T. rangeli was used as an outgroup. The scale bar indicates the number of substitutions per sequence position and the number 00 above the node dividing Lineages and indicates that this topology was supported by 00% bootstrap replicates. This tree inferred by Maximum likelihood has the same topology observed when Parsimony methods were applied but differs from Neighbor Joining trees. In most reconstructions the clade representing group / could not be resolved. (Bas, Basileu strain; Tul, Tulahuen strain). Phylogenetic reconstructions using the SSU gene indicate that divergence between the two T. cruzi lineages is greater than the distances separating four species of Leishmania and comparable to distances among trypanosomatid genera Crithidia, Leishmania, Endotrypanum and Leptomonas (Briones et al. 999). Using patristic distances (sum of branch lengths) of the maximum likelihood tree and the evolutionary rates of 0.85% sequence divergence/00 million years and % sequence divergence/00 million years (Escalante & Ayala 995) we estimated the divergence time of the two lineages to be 88 to 37 million years, respectively.

5 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I, Because the rate of % sequence divergence/00 million years for the SSU rdna might reflect the evolution of faster evoling segments of the molecule we believe that 88 million years of sequence divergence more likely reflects an average rate for the entire gene (Briones et al. 999). CONCLUDING REMARKS Evidence supporting the existence of two major phylogenetic lineages in T. cruzi is accumulating from independent genomic markers, DNA sequence comparison, population genetics and promoter activity from multigenic families. Data indicate that these lineages are distinct evolutionary units. In addition, epidemiological and biological studies suggest that these lineages have different characteristics. Therefore, the division of T. cruzi into two major groups is evident not only genotypically but also physiologically. It is time for the re-evaluation of the T. cruzi taxon and to work out a common nomenclature for these lineages that will serve not only taxonomists but the general community of researchers working with T. cruzi. Further genetic characterization of the subgroups within these lineages is a fundamental step towards the understanding of the complex epidemiological and clinical manifestations of Chagas disease. REFERENCES Brenière SF, Bosseno MF, Telleria J, Bastrenta B, Yacsik N, Noireau F, Alcazar JL, Barnabé C, Wicker P, Tibayrenc M 998. Different behaviour of two Trypanosoma cruzi major clones: transmission and circulation in young bolivian patients. Exp Parasitol 89: Briones MRS, Souto RP, Stolf BS, Zingales B 999. The evolution of two Trypanosoma cruzi subgroups inferred from rrna genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity. Mol Biochem Parasitol (in press). 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